Living-cell imaging of transgenic rat anterior pituitary cells in primary culture reveals novel characteristics of folliculo-stellate cells

Abstract

Folliculo-stellate (FS) cells in the anterior pituitary gland appear to possess multifunctional properties. Recently, the development of transgenic rats (S100b-green fluorescent protein (GFP) rats) that express GFP specifically in FS cells in the anterior pituitary gland has allowed us to distinguish and observe living FS cells in other kinds of pituitary cells. We used S100b-GFP rats to investigate the topographic affinity of FS cells for other pituitary cells. We observed living FS cells in enzymatically dispersed anterior pituitary cells of S100b-GFP rats under a fluorescent microscope, and noted that FS cells markedly extended and contracted cytoplasmic processes and formed interconnections with neighboring FS cells. In addition, FS cells adhered to small clusters of GFPnegative cells, which were primarily hormone-producing cells, and these clusters further aggregated during the course of cytoplasmic contraction. In the presence of laminin, fibronectin, and varying types of collagen, FS cells showed marked changes in shape and specific proliferative activity; however, GFP-negative cells did not. On reverse transcription-PCR analysis and immunohistochemistry, FS cells were shown to express integrin subunits, which are the cell surface receptors for extracellular matrix (ECM). In the anterior pituitary gland, FS cells and the various types of hormoneproducing cells generate a unique topography in the presence of basement membrane components and interstitial collagens. The novel characteristics of FS cells observed in the present study suggest that in the anterior pituitary gland, FS cells play important roles in determining and/or maintaining local cellular arrangement in the presence of ECM components.

The extracellular matrix component laminin promotes gap junction formation in the rat anterior pituitary gland

“…These findings suggest that primary culture of isolated FS cells was appropriate for analyzing FS cell-to-FS cell communication in the absence of the effects of hormoneproducing cells. In a previous study using living-cell imaging of S100b-GFP rat anterior pituitary cells, we first examined whether FS cells respond to ECM components and observed that FS cells in primary culture formed numerous interconnections with neighboring FS cells (Horiguchi et al 2010). In the present study, we observed the behavior of isolated FS cells in the presence of laminin without hormone-producing cells.…”

“…One possibility is that the connexin 43 gene is a downstream target for an outside-in integrin signaling cascade (Spragg & Kass 2005); the other is that laminin, via the integrin signaling cascade, promotes the assembly of cell-to-cell adhesion mediated by E-cadherin before gap junction assembly, thereby up-regulating connexin 43 (Jongen et al 1991, Frenzel & Johnson 1996. FS cells express integrin a3, a6, and b1, which are specific laminin receptors (Horiguchi et al 2010), and E-cadherin (Kikuchi et al 2006). As shown in Fig.…”

“…Using S100b-GFP rat anterior pituitary cells, we found that gap junctional cell-to-cell communication was immunohistochemically reconstructed between FS cells in vitro (Horiguchi et al 2008). Furthermore, using living-cell imaging, we revealed that FS cells under the influence of ECM components may play important roles in determining and/or maintaining local cellular arrangement (Horiguchi et al 2010). The above-mentioned morphological and functional characteristics of FS cells lend weight to the supposition that matricrine action influences cell-to-cell communication within the anterior pituitary gland.…”

Matrix metalloproteinase-9 expression in folliculostellate cells of rat anterior pituitary gland

“…To observe FS cell proliferation, the nucleotide analog 5-bromo-2 0 -deoxyuridine (BrdU; Sigma) was incorporated into primary culture and detected in fixed cells according to previously described procedures (Horiguchi et al 2010). Thirty random fields were imaged per well using a confocal laser microscope with a 60-fold objective lens.…”

“…In a series of experiments that investigated the influence on FS cells of laminin -an ECM component of the basement membrane -we found that FS cells exhibited a motile phenotype and enhanced proliferation and that they established numerous interconnections, which closely resembled their cellular arrangement in vivo (Horiguchi et al 2010(Horiguchi et al , 2011a. The high motility of FS cells on laminin suggested that, to migrate on the ECM, they must express matrix-degrading enzymes.…”